Hypoxia-inducible factor-1alpha expression in experimental cirrhosis: correlation with vascular endothelial growth factor expression and angiogenesis

Angiogenesis progresses together with fibrogenesis during chronic liver injury. Hypoxia-inducible factor-1alpha (HIF-1alpha), a master regulator of homeostasis, plays a pivotal role in hypoxia-induced angiogenesis through its regulation of vascular endothelial growth factor (VEGF). The association between hypoxia, angiogenesis and VEGF expression has been demonstrated in experimental cirrhosis. However, expression of HIF-1alpha has yet to be reported. The aim of this study was to investigate the significance of HIF-1alpha expression during experimental liver fibrosis and the relationships between HIF-1alpha expression, VEGF expression and angiogenesis. Cirrhosis was induced in male Wistar rats by intraperitoneal administration of diethyl nitrosamine (DEN) (100 mg/kg, once a week). The serial sections from liver tissues were stained with anti-HIF-1alpha, anti-VEGF and anti-CD34 antibodies before being measured by light microscopy. Our results showed that HIF-1alpha expression gradually increases according to the severity of fibrosis (p<0.01). Moreover, its expression was found to be correlated with angiogenesis (r=0.916) and VEGF expression (r=0.969). The present study demonstrates that HIF-1alpha might have a role in the development of angiogenesis via regulation of VEGF during experimental liver fibrogenesis and suggests that this factor could be a potential target in the manipulation of angiogenesis in chronic inflammatory diseases of the liver.     

Evidence for cell-specific changes with age in expression of oestrogen receptor (ER) alpha and beta in bone fractures from men and women

Oestrogen is recognized as important for maintaining bone mass in men and women. Oestrogen receptor (ER) alpha and the recently described ER-beta are both expressed in bone cells, but have different affinities for oestrogen agonists and plant oestrogens, which could be important in developing treatments for bone loss in both men and women. It is unclear, however, which isoform predominates in bone; cell type and age may influence their relative expression. The present study has compared ER-alpha and ER-beta expression in serial sections of human fracture callus from males (n = 19, age range 5-72 years) and females (n = 15, age range 3-86 years) by indirect immunoperoxidase. Fracture callus was used as it can be readily obtained from individuals over a wide age range and contains a variety of bone cells. Antibody specificity was confirmed by western blotting and comparison of immunoreactivity in sections of breast tumour and benign prostate hyperplasia. No gender difference in ER expression was found in bone from individuals less than 40 years old. Proliferative chondrocytes were positive for both isoforms, but few larger hypertrophic cells were immunoreactive. ER-alpha and ER-beta were co-expressed in osteoclasts, suggesting that oestrogen may act directly on these cells. Osteoblasts, osteocytes, and mesenchymal cells also expressed both isoforms. In women over 40 years of age, however, relatively fewer biopsies contained osteocytes positive for ER-alpha and ER-beta. Likewise, the proportions of osteoblasts and mesenchymal cells expressing ER-beta were reduced but ER-alpha remained unaffected. In contrast, in men over 40 years, only the proportion of biopsies containing ER-beta-positive mesenchymal cells was lower. In these older men and women, ER-alpha and ER-beta expression was retained by the small proliferative chondrocytes. These results demonstrate that gender, age, and cell type are important determinants of ER isoform expression in skeletal cells.     

N‐acetyl resonances in in vivo and in vitro NMR spectroscopy of cystic ovarian tumors

An unassigned and prominent resonance in the region from δ 2.0–2.1 ppm has frequently been found in the in vivo MR spectra of cancer patients. We demonstrated the presence of this resonance with in vivo MRS in the cyst fluid of a patient with an ovarian tumor. ¹H‐NMRS on the aspirated cyst fluid of this patient confirmed the observation. A complex of resonances was observed between 2.0 and 2.1 ppm. It was also present in 11 additional ovarian cyst fluid samples randomly chosen from our biobank. The resonance complex was significantly more prominent in samples from mucinous tumors than in samples from other histological subtypes. A macromolecule (>10 kDa) was found responsible for this complex of resonances. A correlation spectroscopy (COSY) experiment revealed cross peaks of two different types of bound sialic acid suggesting that N‐glycans from glycoproteins and/or glycolipids cause this resonance complex. In the literature, plasma α‐1 acid glycoprotein (AGP), known for its high content of N‐linked glycans, has been suggested to contribute to the δ 2.0–2.1 spectral region. The AGP cyst fluid concentration did not correlate significantly with the peak height of the δ 2.0–2.1 resonance complex in our study. AGP may be partly responsible for the resonance complex but other N‐acetylated glycoproteins and/or glycolipids also contribute. After deproteinization of the cyst fluid, N‐acetyl‐L ‐aspartic acid (NAA) was found to contribute significantly to the signal in this spectral region in three of the 12 samples. GC‐MS independently confirmed the presence of NAA in high concentration in the three samples, which all derived from benign serous tumors. We conclude that both NAA and N‐acetyl groups from glycoproteins and/or glycolipids may contribute to the δ 2.0–2.1 ppm resonance complex in ovarian cyst fluid. This spectral region seems to contain resonances from biomarkers that provide relevant clinical information on the type of ovarian tumor. Copyright © 2009 John Wiley & Sons, Ltd.     

A quantitative comparison of metabolite signals as detected by in vivo MRS with ex vivo 1H HR‐MAS for childhood brain tumours

¹H MRS provides a powerful method for investigating tumour metabolism by allowing the measurement of metabolites in vivo. Recently, the technique of ¹H high‐resolution magic angle spinning (HR‐MAS) has been shown to produce high‐quality data, allowing the accurate measurement of many metabolites present in unprocessed biopsy tissue. The purpose of this study was to evaluate the agreement between the techniques of in vivo MRS and ex vivo HR‐MAS for investigating childhood brain tumours. Short‐TE (30 ms), single‐voxel, in vivo MRS was performed on 16 paediatric patients with brain tumours at 1.5 T. A frozen biopsy sample was available for each patient. HR‐MAS was performed on the biopsy samples, and metabolite quantities were determined from the MRS and HR‐MAS data using the LCModel? and TARQUIN algorithms, respectively. Linear regression was performed on the metabolite quantities to asses the agreement between MRS and HR‐MAS. Eight of the 12 metabolite quantities were found to correlate significantly (P < 0.05). The four worst correlating metabolites were aspartate, scyllo‐inositol, glycerophosphocholine and N‐acetylaspartate, and, except for glycerophosphocholine, this error was reflected in their higher Cramer–Rao lower bounds (CRLBs), suggesting that low signal‐to‐noise was the greatest source of error for these metabolites. Glycerophosphocholine had a lower CRLB implying that interference with phosphocholine and choline was the most significant source of error. The generally good agreement observed between the two techniques suggests that both MRS and HR‐MAS can be used to reliably estimate metabolite quantities in brain tumour tissue and that tumour heterogeneity and metabolite degradation do not have an important effect on the HR‐MAS metabolite profile for the tumours investigated. HR‐MAS can be used to improve the analysis and understanding of MRS data. Copyright © 2009 John Wiley & Sons, Ltd.     

Gonadotropin treatment restores in vitro interleukin-1beta and tumour necrosis factor-alpha production by stimulated peripheral blood mononuclear cells from patients with idiopathic hypogonadotropic hypogonadism

In the present study, we aimed to investigate the effects of testosterone deficiency and gonadotropin therapy on the in vitro production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) by peripheral blood mononuclear cells (PBMCs) from patients with idiopathic hypogonadotropic hypogonadism (IHH) in order to elucidate the modulatory role of androgen in cytokine production. Fifteen male patients with untreated IHH and 15 age-matched healthy male subjects were enrolled in the study. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), free testosterone (FT), sex hormone binding globulin (SHBG), prolactin, and IL-2 and IL-4 levels were also measured. In unstimulated cultures, IL-1beta and TNF-alpha secretion were not significantly different between patient and control groups. However, after stimulation with lipopolysaccharide (LPS), secretion of IL-1beta and TNF-alpha was significantly higher in cultures from untreated patients with IHH than in control subjects. Mean FSH, LH and FT levels were significantly lower, whereas SHBG, IL-2 and IL-4 levels were significantly higher in patients with IHH compared than in controls. In patients with IHH, FT negatively affected the serum levels of IL-4 and in vitro secretion of IL-1beta and TNF-alpha. In addition, IL-2 and IL-4 affected the in vitro secretion of IL-1beta in a positive manner. Gonadotropin therapy decreased both TNF-alpha and IL-1beta in PBMCs from patients with IHH. The levels of serum IL-2 and IL-4 were also decreased by therapy. In conclusion, in the present study, gonadotropin treatment restored the in vitro production of IL-1beta and TNF-alpha by PBMCs from patients with IHH, suggesting that androgen modulates proinflammatory cytokine production, at least directly through its effects on PBMCs. It seems probable that this effect plays an important role in the immunosuppressive action of androgens.     

ERRα augments HIF‐1 signalling by directly interacting with HIF‐1α in normoxic and hypoxic prostate cancer cells

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up‐regulation of hypoxia‐inducible factor 1 (HIF‐1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen‐related receptor‐α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα‐over‐expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF‐1α protein expression and HIF‐1 signalling. These effects could also be observed in ERRα‐over‐expressing cells grown under normoxia, suggesting that ERRα could function to pre‐adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF‐1α via its AF‐2 domain. A ubiquitination assay showed that this ERRα–HIF‐1α interaction could inhibit ubiquitination of HIF‐1α and thus reduce its degradation. Such ERRα–HIF‐1α interaction could be attenuated by XCT790, an ERRα‐specific inverse agonist, resulting in reduced HIF‐1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF‐1α, suggesting ERRα as a potential therapeutic target for cancer treatment. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Regulation of CCR5 expression and MIP-1alpha production in CD4+ T cells from patients with rheumatoid arthritis

Production of CCR5 expression and MIP-1alpha, a ligand of CCR5, by CD4+ T cells from patients with rheumatoid arthritis (RA) were studied. We analysed further the influence of IL-15 stimulation, CD40/CD40 ligand (CD40L) interaction and CCR5 promotor polymorphism. One hundred and fifty-five RA patients and another 155 age- and sex-matched healthy individuals were enrolled. Peripheral CD4+ and double negative (DN) T cells from patients had lower portions of CCR5, whereas synovial CD4+ and DN T cells showed a much higher CCR5 expression. IL-15 significantly up-regulated the expression of CCR5 on purified CD4+ T cells. CD40L expression on synovial CD4+ T cells was increased greatly in CCR5+ portions by IL-15. MIP-1alpha production by synovial CD4+ T cells was also enhanced by IL-15. Co-culture of CD40 expressing synovial fibroblasts with IL-15-activated synovial CD4+ T cells significantly increased MIP-1alpha production. Expression of CCR5 on patients' CD4+ T cells was not influenced by the promotor polymorphism of CCR5 gene. Taken together, these data suggest CCR5+CD4+ T cells infiltrate the inflamed synovium and IL-15 up-regulates CCR5 and CD40L expression further and enhance MIP-1alpha production in synovial CD4+ T cells. Production of MIP-1alpha by synovial fibroblasts is significantly increased by engagement of CD40 with CD40L. Synovial microenvironment plays a potential role in regulation of CCR5+CD4+ T cells in rheumatoid joints.     

Interferon-beta and adhesion molecules (E-selectin and s-intracellular adhesion molecule-1) are detected in sera from patients with retinal vasculitis and are induced in retinal vascular endothelial cells by Toll-like receptor 3 signalling

Retinal vasculitis is a major component of ocular inflammation that plays a role in retinal tissue damage in patients with idiopathic uveitis and Behçet's disease. Here we show that type 1 interferons (IFN α/β) were not detected in sera from normal individuals but were identified in up to 46% of the sera from retinal vasculitis patients. The predominant form of IFN observed was IFN‐β, which was detected in 39% of Behçet's disease patients and 47% of idiopathic uveitis patients. Seven patients whose sera contained IFN‐β were monitored prospectively. IFN‐β was shown to be present for 6–12 months in all seven of the sera samples tested. Furthermore, the adhesion molecule profile identified in this study was strikingly different when Behçet's and uveitis patient sera were compared to sera from normal controls. Sera from Behçet's disease patients contained significantly elevated levels of the soluble adhesion molecules, sE‐selectin and s‐intracellular adhesion molecule‐1 (sICAM‐1), whereas sera from patients with idiopathic uveitis contained significantly increased sE‐selectin. In vitro studies evaluating the cell source of these cytokines revealed that polyriboinosinic polyribocytidylic acid (poly I:C) activated retinal vascular endothelial cells produce sE‐selectin, sICAM‐1 and IFN‐β. Production of these molecules was inhibited by pretreatment with anti‐Toll‐like receptor 3 (TLR‐3) antibody. In conclusion, IFN‐β, sE‐selectin and sICAM‐1 are elevated in patients with retinal vasculitis and are induced in retinal vascular endothelial cells in vitro by activating the innate immune system through TLR‐3. Further analysis of innate immune signalling may prove to be a novel target for future studies on pathogenic mechanisms and therapeutic approaches in retinal vasculitis.